This proposal aims to address the Provocative Question #7: ?What in vivo imaging methods can be developed to determine and record the identity, quantity, and location of each of the different cell types that contribute to the heterogeneity of a tumor and its microenvironment?? We plan to answer this question in a mouse model of osteosarcoma by developing a multi-fluorescent approach to study the osteosarcoma's architecture and cellular dynamics. Osteosarcoma (OS) OS is the most common non-hematological malignant primary tumor in bone. It is mainly diffused in children and is often very aggressive. About 20% of the young patients diagnosed with OS will develop metastases, most commonly in the lung. For the past 20 years, despite numerous advances in treatment, the survival rate for metastasized OS has not changed (still as low as 20%) and osteosarcoma remains the second leading cause of cancer-related deaths in children and young adults. The lack of a robust in vivo OS model that allows for identification, quantification, localization, and tracing of each of the different cell types that contribute to the heterogeneity of OS and its microenvironment represents a major barrier to the development of novel cell-targeting therapeutics for OS. To overcome this barrier we propose to develop a multi-fluorescent cell stage-specific transgenic mouse model of OS based on the ability to drive the expression of three different fluorescent proteins during different stages of osteoblast differentiation and on the possibility of inducing OS by conditional inactivation of p53 and Rb in the osteoblast lineage. Using this model, by means of intravital microscopy and in vivo flow cytometry, we propose to explore the cellular architecture and cellular dynamics of the OS during development, describing the location of various fluorescent cell populations relative to the OS vasculature and identifying the role that they may have in development of OS lung metastases. We believe that the elucidation of the tumor architecture, the interpretations of the cell dynamics during neoplastic development, and the identification of the cell population responsible for OS metastases will be useful to future studies aimed at designing novel approaches to OS cell-targeting therapy.